Conditions resulting in or from a systemic inflammatory response syndrome (SIRS) are associated with an exaggerated immune response, oxygen free-radical-mediated injury, and tissue perfusion maldistribution. Such conditions include endotoxin shock, septic shock, sepsis, endotoxemia, septicemia, peritonitis, and adult respiratory distress syndrome (ARDS). Current treatment is unsatisfactory. Therapeutic attempts to modify cytokine responses during SIRS-related conditions have focussed on antibodies to the cytokines or cytokine receptor antagonists. These approaches have proven unsuccessful because some level of cytokine response is required for survival from SIRS-related conditions.
Adenosine has been reported to be an endogenous modulator of inflammation by virtue of its effects on stimulated granulocyte function (Cronstein et al., 1986) and on macrophage, lymphocyte and platelet function. Adenosine receptor agonists have been reported to be beneficial in an experimental model of inflammation (Schrier et al., 1990). Adenosine and a related analog have been reported to inhibit in vitro production of the cytokine, tumor necrosis factor alpha (Parmely et al., 1991). Antibodies to TNF-.alpha. have not been shown to alter mortality in sepsis (Abraham et al. 1998, Cohen et al. 1996, Amiot et al. 1997).
Adenosine is an endogenous, ubiquitous molecule that modulates immune function, can suppress or increase free-radical production, and produces vasodilation in regions wherein adenosine is produced in significant quantities.
Adenosine has a short half life (&lt;1 sec) in human blood (Moser et al., 1989), and therefore high doses of adenosine would need to be administered continuously to achieve effective treatment levels. Adenosine has been reported to exhibit negative inotropic, chronotropic and dromotropic effects (Belardinelli et al., 1989) and to cause coronary steal by preferentially dilating vessels in nonischemic regions. Consequently, high doses of adenosine are toxic and this toxicity severely limits its therapeutic potential. However, by increasing adenosine concentration locally, i.e. at the target site within the target tissue, the beneficial effects of adenosine might be provided without the toxic systemic effects.
Riches et al. (1985) reported that adenosine inhibited .beta.-galactosidase secretion from zymosan particle-stimulated mouse peritoneal macrophages. The adenosine nucleotides ATP, ADP, and AMP were also effective inhibitors, but only after hydrolysis to adenosine. These authors found that the inhibitory effect of adenosine in vitro could be increased with erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA), a potent inhibitor of adenosine deaminase. By thus inhibiting adenosine breakdown to inosine and hypoxanthine the inhibitory effects of adenosine were prolonged. Similarly, Itoh et al. (1989) reported that both adenosine and 1-methyladenosine inhibited chemiluminescence by zymosan-stimulated mouse peritoneal macrophages in vitro.
Adenosine has been shown to inhibit TNF-.alpha. produced in response to endotoxin (LPS). Using LPS, Eigler et al. stimulated isolated human peripheral blood mononuclear cell production of TNF-.alpha.. The addition of adenosine deaminase (increasing endogenous adenosine degradation) or an adenosine A.sub.2 receptor antagonist further increased TNF-.alpha. production, while an adenosine A.sub.1 receptor antagonist had no effect. This indicated that endogenous adenosine production after stimulation with LPS served to limit the TNF-.alpha. response of the monocyte. Eigler et al. further demonstrated that TNF-.alpha. production by LPS-stimulated monocytes could be inhibited by dipyridamole, an agent that prevents cellular adenosine reuptake a major pathway for adenosine removal by monocytes (Barankiewicz, 1989). Adenosine-modulated TNF-.alpha. production by other cell types has also been shown. Cronstein et al. examined leukocyte accumulation and TNF-.alpha. production in skin air pouches injected with carrageenan. Endogenous adenosine concentrations were altered by inhibiting adenosine kinase, an enzyme contributing to nucleotide salvage via phosphorylation of adenosine. Pre-treatment of rats with oral GP1-515, an adenosine kinase inhibitor (reducing adenosine salvage into nucleotides), reduced leukocyte accumulation and TNF-.alpha. production. TNF-.alpha. concentration in the pouch exudates were reduced from 1518 pg/ml to 780 pg/ml. The direct involvement of adenosine in this response was proven by reversing the inhibitory effects of GP-1-515 with either excess exogenous adenosine deaminase or an adenosine A.sub.2 receptor antagonist.
An adenosine kinase inhibitor, GP-1-515, produced by Gensia Inc., is reported to elevate local adenosine concentrations in tissues. Adenosine deaminase is a cytosolic and membrane-bound enzyme which catalyzes the deamination of adenosine to inosine, a necessary step prior to entry of adenosine catabolites into the xanthine oxidase pathway to form uric acid. Inhibition of adenosine deaminase can reduce the rate at which extracellular adenosine is degraded, leading to increased adenosine outside of the cell where it is pharmacologically active. Inhibition of ADA has such an effect. In isolated guinea pig hearts addition of the adenosine deaminase inhibitor, EHNA, to the perfusion medium, in the presence of 5'-amino-5'-deoxyadenosine to inhibit phosphorylation of adenosine to AMP, was reported to result in a 15-fold increase of adenosine release (Schrader, 1983). These effects were not apparent in the absence of ADA inhibition.
In an effort to find effective treatments for SIRS and related conditions, inhibitors of adenosine deaminase were explored.